High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer |
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Authors: | Dietmar Reusch Markus Haberger Tobias Kailich Anna-Katharina Heidenreich Michael Kampe Patrick Bulau Manfred Wuhrer |
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Institution: | 1.Pharma Biotech Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany;2.Center for Proteomics and Metabolomics; Leiden University Medical Center; Leiden, The Netherlands;3.Division of BioAnalytical Chemistry; Department of Chemistry and Pharmaceutical Sciences; VU University Amsterdam; Amsterdam, The Netherlands |
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Abstract: | The Fc glycosylation of therapeutic antibodies is crucial for their effector functions and their behavior in pharmacokinetics and pharmacodynamics. To monitor the Fc glycosylation in bioprocess development and characterization, high-throughput techniques for glycosylation analysis are needed. Here, we describe the development of a largely automated high-throughput glycosylation profiling method with multiplexing capillary-gel-electrophoresis (CGE) with laser induced fluorescence (LIF) detection using a DNA analyzer. After PNGaseF digestion, the released glycans were labeled with 9-aminopyrene-1,3,6-trisulfonic acid (APTS) in 96-well plates, which was followed by the simultaneous analysis of up to 48 samples. The peak assignment was conducted by HILIC-UPLC-MS/MS of the APTS-labeled glycans combined with peak fractionation and subsequent CGE-LIF analysis of the MS-characterized fractions. Quantitative data evaluation of the various IgG glycans was performed automatically using an in-house developed software solution. The excellent method accuracy and repeatability of the test system was verified by comparison with two UPLC-based methods for glycan analysis. Finally, the practical value of the developed method was demonstrated by analyzing the antibody glycosylation profiles from fermentation broths after small scale protein A purification. |
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Keywords: | monoclonal antibody (mAb) IgG glycosylation automation multiplexed capillary electrophoresis DNA analyzer HILIC-UPLC APTS labeling LC-MS |
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